Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other country.
Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.
Cytokines control a wide variety of biological responses, thus the duration and intensity of their effects must be tightly regulated. Upon stimulation by cytokine, specific cell surface receptors oligomerise and cause activation of the JAK-STAT signaling pathway (Heim, 1999; Ihle et al., 1998; Leonard and O'Shea, 1998). The transient nature of this signaling cascade is partly a consequence of the action of negative regulatory molecules such as SHP-1, PIAS-3 and the SOCS (suppressor of cytokine signaling) (alternate names SSI, JAB, CIS) proteins, each of which inhibit the JAK-STAT signaling pathway and ensure the appropriate level of response to a particular cytokine stimulus is maintained (Endo et al., 1997; Gissclbrecht, 1999; Hilton, 1999; Naka et al., 1997; Starr et al., 1997; Yoshimura, 1998).
The SOCS family of proteins comprises a number of members, including SOCS-1 through SOCS-7 and CIS, and the expression of several of these is known to be induced by cytokines (Hilton, 1999; Hilton et al., 1998; Yoshimura, 1998). SOCS-7 and CIS contain two regions of homology—a central SH2 domain and a C-terminal 40 amino acid motif known as the SOCS box. While the SOCS box acts to recruit elongins BC, a protein complex implicated in the proteasomal degradation pathway (Kamura et al., 1998; Zhang et al., 1999), the SH2 domains of the SOCS proteins are responsible for specific binding to activated (phosphorylated) signaling molecules and may also play a role in the mechanism of signal suppression. For instance, CIS binds via its SH2 domain to phosphorylated erythropoietin (EPO) and interleukin-3 (IL-3) receptors at the same sites used for STAT-5 binding, thus preventing docking and activation of this transcription factor (Matsumoto et al., 1997; Yoshimura et al., 1995). By contrast, the SH2 domain of SOCS-1 binds to activated JAKs, and together with the action of an additional protein interaction motif upstream of the SH2 domain, results in inhibition of the kinase catalytic activity (Nicholson et al., 1999; Sasaki et al., 1999).
A number of studies have identified cytokines which can induce the expression of SOCS-3 mRNA, including CNTF (Bjorback et al., 1999), LIF (Bousquet et al., 1999), IL-6 (Starr et al., 1997), IL-11 (Auernhammer and Melmed, 1999a), leptin (Bjorbaek et al., 1998), IL-2 (Cohney et al., 1999), IL-10 (Donnelly et al., 1999; Ito et al., 1999), prolactin (Pezet et a!., 1999), growth hormone (Adams et al., 1998) and insulin. Overexpression of SOCS-3 results in the inhibition of signaling by each of these cytokines, and under these conditions SOCS-3 has been shown to physically associate with either JAK kinase (Sasaki et al., 1999), or the receptors for growth hormone (Hansen et al., 1999), IL-2Rβ (Cohney et al., 1999), and insulin receptors. However, given that overexpression can lead to elevated protein levels at which non-specific interactions can result, it is difficult to assess whether all of these observations have a genuine biological relevance. Alternatively, gene knockout studies have shown that SOCS-3−/− mice die embryonically from a disease possibly associated with excessive foetal erythropoiesis (Marine et al., 1999).
Recently, it was proposed that the mechanism by which SOCS-3 inhibits signaling is identical to that of SOCS-1. As had been demonstrated for SOCS-1 (Yasukawa et al., 1999), SOCS-3 was also shown to associate with JAK2 in intact cells, and to a synthetic phosphopeptide encompassing the activation loop from JAK2 (Sasaki et al., 1999). The region of SOCS-3 immediately N-terminal to the SH2 domain has also been shown to be important for biological activity (Narazaki et al., 1998; Nicholson et al., 1999; Sasaki et al., 1999), and based on sequence similarity to SOCS-1, was also proposed to function as a kinase active site inhibitor. However, despite these similarities, there is evidence to suggest that the mechanism of signaling suppression used by SOCS-3 differs from that of SOCS-1. Unlike SOCS-1, SOCS-3 does not inhibit the catalytic activity of JAK1 or JAK2 in an in vitro kinase reaction (Nicholson et al., 1999). Furthermore, the kinetics of IL-6 signal suppression, as measured by inhibition of STAT3 phosphorylation, is considerably slower for SOCS-3 as compared to SOCS-1 (Suzuki et al., 1998). While forced expression of SOCS-1 in M1 cells results in rapid and total inhibition of STAT3 phosphorylation within 30 minutes, inhibition by SOCS-3 slowly increases over the course of several hours (Suzuki et al., 1998). These differences between SOCS-1 and SOCS-3 have been explained on the basis of a weaker affinity of SOCS-3 for JAK kinase (Masuhara et al., 1997; Sasaki et al., 1999). However, another possible explanation is that the primary binding target for SOCS-3 is not the JAK kinase, but other molecules within the signaling cascade such as the phosphorylated cytokine receptors or STAT proteins.
In work leading up to the present invention, the inventors sought to identify the molecular target of SOCS-3 and quantify the affinity of this interaction. Based on peptide binding data, the subject inventors have demonstrated that a single high affinity binding site exists for SOCS-3 on the gp130 receptor, centred around phosphotyrosine-757 (pY757). The letter “p” before a single letter abbreviation for an amino acid (e.g. “Y” for tyrosine) means the amino acid is phosphorylated. The numerical value after the amino acid letter is the residue number. Binding is phosphorylation dependent, and suppression of gp130 signaling by SOCS-3, but not SOCS-1, is impaired if this residue is mutated to phenylalanine. Furthermore, SOCS-3 binds to a gp130-derived pY757 phosphopeptide with an affinity that is approximately 104-fold higher than binding to a phosphopeptide derived from the activation loop in JAK2, previously reported to be the biologically relevant docking site for SOCS-3 (Sasaki et al., 1999).
Taken together, these data show that suppression of gp130-mediated signaling by SOCS-3 involves recruitment to the phosphorylated receptor in a manner that is distinct to the mechanism of inhibition used by SOCS-1. These data also have similar implications in relation to other cytokine receptors such as erythropoietin (EPO) receptor and leptin receptor which have phosphotyrosine regions homologous to those recognized by SOCS-3 on gp130.